Information is available about the overall mechanism of mitochondrial DNA (mtDNA) replication but not about the details. These can be elucidated only by enzymatic studies on the mtDNA polymerase and on auxiliary enzymes and proteins which must be used in concert with the polymerase in reconstitution experiments. Our laboratory has been isolating such mitochondrial enzymes, namely, a DNA polymerase and two topoisomerases (type I, capable of relaxing supercoiled DNA, and type II, capable of relaxing, catenating, and decatenating mtDNA); others have isolated a single strand binding protein. While our laboratory has obtained indirect evidence of a super-coiling enzyme in intact mitochondria, no gyrase is detectable in extracts. Present Aims: (1) To study the action of mtDNA polymerase on its natural template, mtDNA; heretofore only nuclear DNA or homopolymers were used. The assay would be the extent of extension of the D loop strand (the short nascent daughter strand in replicating mtDNA). (2) To prepare adequate amounts of various primer templates by cloning into a vector the entire mtDNA molecule and various segments containing the D loop and using the hybrid molecules as comparative templates. The primer would be obtained by excising the cloned D loop region and separating the complementary strands, and by chemical synthesis. Introduction of these strands into mtDNA to form a D loop occurs as a spontaneous reaction. (3) To determine how the replication system overcomes the topological constraints imposed by unwinding of the double helix. a) The effect of the topoisomerases on the maximum possible extension of the D loop strand will be determined. b) The question will be asked whether this extension leads to the generation of positive supercoils, or alternatively, whether it never goes any farther, or never even reaches the point of DNA relaxation, suggesting the necessity of periodic generation of negative supercoils. (4) To study priming activity at the H strand replication origin (located in the D loop region). Primase activity will be sought in fractionated mitochondrial extracts as well as in the known mtDNA polymerase and will be assayed by D loop strand formation in the presence of mtDNA polymerase. (5) To seek other enzymes/proteins which play a role in replication. mtDNA polymerase would be used in concert with mitochondrial extracts fractionated in novel ways. (6) To investigate the final steps in replication, including a) isolation of a mitochondrial ligase, and b) testing the type II topoisomerase for its ability to separate the two daughter circles.